Anti-ricin antibodies and uses thereof

ABSTRACT

The present invention relates to anti-ricin antibodies and uses thereof. More specifically, the invention relates to anti-ricin antibodies and fragments thereof as well as their use in therapy or prophylaxis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 14/122,366filed Nov. 26, 2013, which is a national entry of International PatentApplication PCT/CA2012/000092 filed Jan. 31, 2012 and claims the benefitof United States Provisional Patent Application U.S. Ser. No. 61/495,544filed Jun. 10, 2011, the entire contents of all of which are hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to anti-ricin antibodies and uses thereof.More specifically, the invention relates to anti-ricin antibodies andfragments thereof as well as their use in therapy or prophylaxis.

BACKGROUND OF THE INVENTION

Ricin is a 60-65 kDa glycoprotein derived from beans of the castor plant(Montanaro et al, 1973). It is a relatively simple toxin consisting of aricin toxin enzymatic-A (RTA) protein and a ricin toxin lectin-B (RTB)protein linked by a disulfide bond. The RTB is responsible for bindingto specific sugar residues on the target cell surface and allowsinternalizaition of ricin by endocytosis, whereas the RTA enzymaticallyinactivates the ribosome to irreversibly inhibit protein synthesis. Asingle molecule of RTA within the cell can completely inhibit proteinsynthesis, resulting in cell death. Ricin is one of the most potenttoxins known for humans, with an LD₅₀ of 1-20 mg/kg body weight wheningested and 1-20 μg/kg when inhaled or injected (Audi et al, 2005);this is 400 times more toxic than cobra venom, 1000 times more toxicthan cyanide, and 4000 times more toxic than arsenic. Ricin is listed onthe Centers for Disease Control and Prevention (Atlanta, USA) Category Bthreat list and is regarded as a high terrorist risk for civilians.Unfortunately, there is currently no therapeutic or vaccine availableagainst ricin.

The development of therapeutics against ricin has proven elusive.Chemical inhibitors targeting ricin have been developed, but these arelimited by the high amounts needed for short-term effects and their owntoxicity (Burnett et al, 2005; Miller et al, 2002). Development ofvaccines against ricin is ongoing, but to date such vaccines have onlypartially protected mice against ricin (Smallshaw et al, 2007). Of thedifferent approaches for medical countermeasures, the development ofanti-ricin antibodies appears the most promising. Much work has beendone on developing antibodies, both polyclonal and monoclonal, astherapeutics against the toxin.

These antibodies were directed against the toxic A-chain (blocking itsdestructive action to the ribosome) or the lectin B-chain (preventing itfrom binding to and entering the cell). (Neal et al, 2010; Foxwell B M Jet al, 1985)

A sheep anti-ricin F(ab)₂ was developed in the United Kingdom forresearch and development as well as for potential emergency use.However, large amounts, about 50-100 μg of polyclonal antibodies (pAbs)(Neal et al, 2010) or 5-100 μg of mAbs (Hewetson et al, 1993; Foxwell etal, 1985), are needed either to protect or treat a mouse from ricinpoisoning within a small window of time, providing significantlimitations on survival. For example, 5 μg antibody delivered by theintra-peritoneal (i.p.) route had to be given within 24 h to protectmice before 5×LD₅₀ ricin challenge (Neal et al, 2010), while 100 μg ofmAb per mouse had to be given within 30 min after 10×LD₅₀ ricinchallenge (Guo et al, 2006).

It was previously reported that mice could be immunized using increasingdoses of ricin, their spleens harvested, and hybridoma created by fusingthe lymphocytes with myeloma cells (Furukawa-Stoffer et al, 1999). Apoisoning method was then used to select clones that survived in culturemedium with ricin because these secreted sufficient amounts ofanti-ricin neutralizing mAbs. The antibodies from these clones had highneutralizing activity against ricin, as judged by their binding to thetoxin in an enzyme linked immunosorbent assay (ELISA) and by ricinneutralization experiments. HRF4 was identified as the best mAb.

While HRF4 showed promising activity in previous studies, there remainsa need in the art for highly effective molecules for neutralization ofricin activity. Such molecules would be advantageous in the developmentof medical countermeasure therapy.

SUMMARY OF THE INVENTION

The present invention relates to anti-ricin antibodies and uses thereof.More specifically, the invention relates to anti-ricin antibodies andfragments thereof as well as their use in therapy or prophylaxis.

The present invention provides an isolated or purified antibody orfragment thereof, comprising

-   -   a variable light chain comprising        -   the sequence of complementarity determining region (CDR) L1            selected from sequences KASQDIKQYIA (SEQ ID NO:1),            KASQDINNYLR (SEQ ID NO:2), KASQDIKKYIG (SEQ ID NO:3), and            KASQDVTAAVA (SEQ ID NO:4);        -   the sequence of CDR L2 selected from sequences YTSTLQP (SEQ            ID NO:5), RANRLVD (SEQ ID NO:6), YTSTLQP (SEQ ID NO:7), and            SASYRYT (SEQ ID NO:8); and        -   the sequence of CDR L3 selected from sequences LQYDHLYT (SEQ            ID NO:9), LQYDEFPYT (SEQ ID NO:10), LQYDSLYT (SEQ ID NO:11),            and QQYYNTPLT (SEQ ID NO:12), and    -   a variable heavy chain comprising        -   the sequence of complementarity determining region (CDR) H1            selected from sequences SYWIQ (SEQ ID NO:13), EYIIN (SEQ ID            NO:14), NYWIE (SEQ ID NO:15), and EHIIN (SEQ ID NO:16);        -   the sequence of CDR H2 selected from sequences            EILPGTGNTNYSEKFKG (SEQ ID NO:17), WFYPGSGDIKYNEKFKD (SEQ ID            NO:18), EILPGSGSINYDEKFKG (SEQ ID NO:19), and            LINPNSGGTNYNQKFKD (SEQ ID NO:20); and        -   the sequence of CDR H3 selected from sequences CEGEGYFQAWFAY            (SEQ ID NO:21), NGRWDDDYFDY (SEQ ID NO:22), QANRGFDSAWFAY            (SEQ ID NO:23), and LRYDAAY (SEQ ID NO:24),

wherein the antibody or fragment thereof specifically recognizes andbinds to ricin.

The isolated or purified antibody or fragment thereof as described abovemay comprise a variable chain comprising a CDR L1 of sequenceKASQDIKQYIA (SEQ ID NO:1), a CDR L2 of sequence YTSTLQP (SEQ ID NO:5),and a CDR L3 of sequence LQYDHLYT (SEQ ID NO:9); and a variable heavychain comprising CDR H1 of sequence SYWIQ (SEQ ID NO:13), a CDR H2 ofsequence EILPGTGNTNYSEKFKG (SEQ ID NO:17), and a CDR H3 of sequenceCEGEGYFQAWFAY (SEQ ID NO:21).

In another example, the isolated or purified antibody or fragmentthereof may comprise a variable chain comprising a CDR L1 of sequenceKASQDINNYLR (SEQ ID NO:2), a CDR L2 of sequence RANRLVD (SEQ ID NO:6),and a CDR L3 of sequence LQYDEFPYT (SEQ ID NO:10); and a variable heavychain comprising CDR H1 of sequence EYIIN (SEQ ID NO:14), a CDR H2 ofsequence WFYPGSGDIKYNEKFKD (SEQ ID NO:18), and a CDR H3 of sequenceNGRWDDDYFDY (SEQ ID NO:22).

In a further example, the isolated or purified antibody or fragmentthereof as described above may comprise a variable chain comprising aCDR L1 of sequence KASQDIKKYIG (SEQ ID NO:3), a CDR L2 of sequenceYTSTLQP (SEQ ID NO:7), and a CDR L3 of sequence LQYDSLYT (SEQ ID NO:11);and a variable heavy chain comprising CDR H1 of sequence NYWIE (SEQ IDNO:15), a CDR H2 of sequence EILPGSGSINYDEKFKG (SEQ ID NO:19), and a CDRH3 of sequence QANRGFDSAWFAY (SEQ ID NO:23).

In an alternative example, the isolated or purified antibody or fragmentthereof of as described above may comprise a variable chain comprising aCDR L1 of sequence KASQDVTAAVA (SEQ ID NO:4), a CDR L2 of sequenceSASYRYT (SEQ ID NO:8), and a CDR L3 of sequence QQYYNTPLT (SEQ IDNO:12); and a variable heavy chain comprising CDR H1 of sequence EHIIN(SEQ ID NO:16), a CDR H2 of sequence LINPNSGGTNYNQKFKD (SEQ ID NO:20),and a CDR H3 of sequence LRYDAAY (SEQ ID NO:24).

In yet a further alternative, the isolated or purified antibody orfragment thereof as described above may comprise a variable light chainsequence selected from:

(SEQ ID NO: 25) DIQMTQSPSSLSASLGGKVTITCKASQDIKQYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLDPEDIATYYCLQYDHLYTFGGG TKLEIKR; (SEQ ID NO:27) DIVLTQSPSSMYASLGERVTITCKASQDINNYLRWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGFYSCLQYDEFPYTFGG GTKLEIKR; (SEQ ID NO:29) DIQMTQSPSSLSAFVGGKVTITCKASQDIKKYIGWYQQKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDSLYTFGGG TKLEIKR; (SEQ ID NO:31) DIELTQSHKFMSTSVGDRVSITCKASQDVTAAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGSDFTFTISSVQAEDLAVYYCQQYYNTPLTFGA GTKLELKR;and

-   -   a sequence substantially identical thereto

and a variable heavy chain sequence selected from:

(SEQ ID NO: 26) KVQLQESGAELMKPGASVKISCKATGYTFSSYWIQWIKQRPGHGLEWIGEILPGTGNTNYSEKFKGKATFTTDTSSNTAYMHFSSLTSEDSAVYYCSRCEGEGYFQAWFAYWGQGTTVTVSS; (SEQ ID NO: 28)EVQLQESGTGLVKPGASVKLSCKASGYTFTEYIINWVKQRSGQGLEWIGWFYPGSGDIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARNG RWDDDYFDYWGQGTTVTVSS;(SEQ ID NO: 30) KVKLQESGAELMKPGASVKISCKSTGYTFSNYWIEWIKQRPGHGLEWIGElLPGSGSINYDEKFKGKATFTADTSSDTVYMFLSGLTSEDSAVYYCARQANRGFDSAWFAYWGQGTTVTVSS; (SEQ ID NO: 32)QVQLQESGPELVKPGASMKISCKASGYSFTEHIINWVKQTHRENLEWIGLINPNSGGTNYNQKFKDKATLTVDTASNTAYMELLSLTSEDSAVYYCARLR YDAAYWGQGTTVTVSS;and

-   -   a sequence substantially identical thereto.

The isolated or purified antibody or fragment thereof as described byany of the above may comprise:

the variable light chain sequence:

(SEQ ID NO: 25) DIQMTQSPSSLSASLGGKVTITCKASQDIKQYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLDPEDIATYYCLQYDHLYTFGGG TKLEIKR

and the variable heavy chain sequence:

(SEQ ID NO: 26) KVQLQESGAELMKPGASVKISCKATGYTFSSYWIQWIKQRPGHGLEWIGEILPGTGNTNYSEKFKGKATFTTDTSSNTAYMHFSSLTSEDSAVYYCSRCEGEGYFQAWFAYWGQGTTVTVSS;or

the variable light chain sequence:

(SEQ ID NO: 27) DIVLTQSPSSMYASLGERVTITCKASQDINNYLRWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGFYSCLQYDEFPYTFGG GTKLEIKR

and the variable heavy chain sequence:

(SEQ ID NO: 28) EVQLQESGTGLVKPGASVKLSCKASGYTFTEYIINWVKQRSGQGLEWIGWFYPGSGDIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARNG RWDDDYFDYWGQGTTVTVSS;or

the variable light chain sequence:

(SEQ ID NO: 29) DIQMTQSPSSLSAFVGGKVTITCKASQDIKKYIGWYQQKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDSLYTFGGG TKLEIKR

and the variable heavy chain sequence:

(SEQ ID NO: 30) KVKLQESGAELMKPGASVKISCKSTGYTFSNYWIEWIKQRPGHGLEWIGEILPGSGSINYDEKFKGKATFTADTSSDTVYMFLSGLTSEDSAVYYCARQANRGFDSAWFAYWGQGTTVTVSS;or

the variable light chain sequence:

(SEQ ID NO: 31) DIELTQSHKFMSTSVGDRVSITCKASQDVTAAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGSDFTFTISSVQAEDLAVYYCQQYYNTPLTFGA GTKLELKR

and the variable heavy chain sequence:

(SEQ ID NO: 32) QVQLQESGPELVKPGASMKISCKASGYSFTEHIINWVKQTHRENLEWIGLINPNSGGTNYNQKFKDKATLTVDTASNTAYMELLSLTSEDSAVYYCARLR YDAAYWGQGTTVTVSS,

or a sequence substantially identical thereto.

The isolated or purified antibody or fragment thereof of the presentinvention may be specific for the ricin toxin lectin-B protein. Theisolated or purified antibody or fragment thereof of may be an IgG.

The present invention also provides a nucleic acid sequence encoding theisolated or purified antibody or fragment thereof as described herein.The invention also encompasses a vector comprising the nucleic acidmolecule just described, and hybridoma cell lines expressing theisolated or purified antibody or fragment thereof described above.

The present invention additionally provides a composition comprising oneor more than one antibody or fragment thereof of the present inventionand a pharmaceutically acceptable diluent, excipient, or carrier. Thecomposition may be a vaccine composition.

The present invention further provides a method of preventingdeleterious effects caused by ricin exposure or of treating exposure toricin, comprising administering one or more than one antibody orfragment thereof or the composition of the present invention to asubject in need thereof. The subject in need thereof may be a mammal,such as a mouse or a human.

In the method as described above, the one or more than one antibody orfragment thereof or composition comprising same may be administered tothe subject several hours following exposure to the ricin toxin to treatricin exposure. Alternatively, or in addition, the one or more than oneantibody or fragment thereof or composition thereof may be administeredto the subject several weeks prior to exposure to the ricin toxin toprotect the subject against ricin exposure.

Additionally, a combination of antibodies or fragments thereof of thepresent invention may provide a synergistic effect on ricin-neutralizingactivity in the methods as just described. One of the antibodies orfragments thereof may be mAb D9 or a fragment thereof; the secondantibody or fragment thereof may be mAb B10 or a fragment thereof.

The present invention further encompasses a method of conferringimmunity against ricin comprising administering one or more than oneantibody or fragment thereof or a composition of the present inventionto a subject in need thereof.

Additionally, the present invention provides a method of identifyinghybridoma secreting effective anti-ricin antibodies, comprising:

-   -   a) providing hybridoma cells prepared from lymphocytes obtained        from mice immunized against ricin;    -   b) exposing the cells to high amounts of ricin; and    -   c) identifying the cells that survive exposure step b).

In the method as just described, the mice from which the splenocytes areobtained may have been immunized using multiple lethal doses of ricin.In the above method, the high amount of ricin used in step b) may be inthe range of 1 to 10 ng/ml or 1 to 5 ng/ml.

Four hybridoma clones were developed that secreted high-titre anti-ricinIgG antibodies. These mAbs have great potential to be developed asantibody-based therapeutic agents or antibody-gene based vaccinesagainst ricin. All four mAbs were found to have highricin-neutralization potency both in an in vitro neutrallization assayand an in vivo antibody/ricin co-incubation assay, indicating the stronginhibition of ricin-mediated cell death. Monoclonal antibody D9, foundto be exceptionally active in the mouse assay, was further tested forpost-exposure therapy and pre-exposure prophylaxis against ricin invivo. It protected mice not only hours, but also several weeks (at least6 weeks) before toxin challenge (5×LD₅₀ of ricin), and rescued mice upto 6 hours after poisoning (5×LD₅₀ of ricin); additionally, low amounts(0.5 μg) were therapeutic against high amounts of toxin (1 μg of ricin).Antibody D9 also showed synergistic effects with other anti-ricin mAb,as determined by the in vitro neutralization assay. A dose of 5 μgantibody in a mouse is equivalent to 1.4 mg in a human. These resultsindicate that milligram amounts of specific anti-ricin monoclonalantibody in very small volumes (0.1 ml) may be sufficient to protectfirst responders or treat ricin-exposed casualties.

Additional aspects and advantages of the present invention will beapparent in view of the following description. The detailed descriptionand examples, while indicating preferred embodiments of the invention,are given by way of illustration only, as various changes andmodifications within the scope of the invention will become apparent tothose skilled in the art in light of the teachings of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by wayof example, with reference to the appended drawings, wherein:

FIG. 1 is a bar graph showing the immunoreactivity of the monoclonalantibodies of the present invention. ELISA experiments were performed onindividual antibodies at varying dosages. All the mAb (A9, B10, D3, andD9) bound to ricin in a dose-dependent manner. HRF4 was used as apositive control. The absorbance was read at 615 nm.

FIG. 2 is a Western blot of monoclonal antibody B10 against ricin todetermine the general specificity of the antibody. Lane 1—ricin inreducing conditions (2.2 μg/lane); Lane 2—ricin (1.1 μg/lane); Lane3—ricin A chain (0.4 μg/lane); Lane 4—ricin B chain (0.4 μg/lane);M—molecular weight markers.

FIG. 3 is a graph depicting the half-life of D9 in mouse serum. D9 atthe dose of 5 μg was administered by the i.p. route into mice. Mice weresacrificed at different time points to calculate plasma concentration ofD9 using an immunoassay. The D9 remaining in sera is expressed aspercentages plotted against time in days on the figure.

FIG. 4 is a bar graph depicting the effect of combining mAb of thepresent invention. Antibodies were mixed at a 1:1 ratio (totalconcentration 313 ng/ml) and assayed in vitro using Amalar Blue dye. Asynergistic effect was noted when mAb D9 was combined with otherantibodies of the present invention.

FIG. 5 depicts humanization of mouse D9 Fv by CDR-grafting. Residues arenumbered according to Kabat. CDRs are marked with unshaded boxes. Key FRresidues are marked with *. Two key FR residues in D9 VH, which are keptin hD9 VH are marked with shaded boxes. VH D9 (SEQ ID N0:32); VH 1-18(SEQ ID N0:37); VH JH6 (SEQ ID N0:38); VH hD9 (SEQ ID NO:41); VL D9 (aa1-107 of SEQ ID NO:31); VL 012 (SEQ ID N0:39); VL Jk4 (SEQ ID N0:40) andVL hD9 (SEQ ID N0:42).

FIG. 6 depicts a schematic diagram of the hD9 gene layout.

FIG. 7 SDS-PAGE analysis of purified hD9. Samples were resolved bySDS-PAGE. Lane 1 is a molecular marker; lanes 2 and 4 are control humanIgG1 and hD9 in non-reducing conditions; lanes 3 and 5 are control humanIgG1 and hD9 in reducing conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to anti-ricin antibodies and uses thereof.More specifically, the invention relates to anti-ricin antibodies andfragments thereof as well as their use in therapy or prophylaxis.

The present invention is directed to anti-ricin antibodies and fragmentsthereof. The present invention also covers methods of obtaining andidentifying antibodies specific for and effective against ricin. Thepresent invention further includes methods of using the anti-ricinantibodies of the invention in anti-ricin therapy and prophylaxis.

The present invention provides an isolated or purified antibody orfragment thereof specific to ricin, comprising

a variable light chain comprising

-   -   the sequence of complementarity determining region (CDR) L1        selected from sequences KASQDIKQYIA (SEQ ID N0:1), KASQDINNYLR        (SEQ ID N0:2), KASQDIKKYIG (SEQ ID N0:3), and KASQDVTAAVA (SEQ        ID N0:4);    -   the sequence of CDR L2 selected from sequences YTSTLQP (SEQ ID        N0:5), RANRLVD (SEQ ID N0:6), YTSTLQP (SEQ ID N0:7), and SASYRYT        (SEQ ID N0:8); and    -   the sequence of CDR L3 selected from sequences LQYDHLYT (SEQ ID        N0:9), LQYDEFPYT (SEQ ID N0:10), LQYDSLYT (SEQ ID N0:11), and        QQYYNTPLT (SEQ ID N0:12), and

a variable heavy chain comprising

-   -   the sequence of complementarity determining region (CDR) H1        selected from sequences SYWIQ (SEQ ID NO:13), EYIIN (SEQ ID        NO:14), NYWIE (SEQ ID NO:15), and EHIIN (SEQ ID NO:16);    -   the sequence of CDR H2 selected from sequences EILPGTGNTNYSEKFKG        (SEQ ID NO:17), WFYPGSGDIKYNEKFKD (SEQ ID NO:18),        EILPGSGSINYDEKFKG (SEQ ID NO:19), and LINPNSGGTNYNQKFKD (SEQ ID        NO:20); and    -   the sequence of CDR H3 selected from sequences CEGEGYFQAWFAY        (SEQ ID NO:21), NGRWDDDYFDY (SEQ ID NO:22), QANRGFDSAWFAY (SEQ        ID NO:23), and LRYDAAY (SEQ ID NO:24).

The term “antibody”, also referred to in the art as “immunoglobulin”(Ig), used herein refers to a protein constructed from paired heavy andlight polypeptide chains; various Ig isotypes exist, including IgA, IgD,IgE, IgG, and IgM. When an antibody is correctly folded, each chainfolds into a number of distinct globular domains joined by more linearpolypeptide sequences. For example, the immunoglobulin light chain foldsinto a variable (V_(L)) and a constant (C_(L)) domain, while the heavychain folds into a variable (V_(H)) and three constant (C_(H), C_(H2),C_(H3)) domains. Interaction of the heavy and light chain variabledomains (V_(H) and V_(L)) results in the formation of an antigen bindingregion (Fv). Each domain has a well-established structure well-known tothose of skill in the art.

The light and heavy chain variable regions are responsible for bindingthe target antigen and can therefore show significant sequence diversitybetween antibodies. The constant regions show less sequence diversity,and are responsible for binding a number of natural proteins to elicitimportant biochemical events. The variable region of an antibodycontains the antigen binding determinants of the molecule, and thusdetermines the specificity of an antibody for its target antigen. Themajority of sequence variability occurs in six hypervariable regions,three each per variable heavy and light chain; the hypervariable regionscombine to form the antigen-binding site, and contribute to binding andrecognition of an antigenic determinant. The specificity and affinity ofan antibody for its antigen is determined by the structure of thehypervariable regions, as well as their size, shape, and chemistry ofthe surface they present to the antigen. Various schemes exist foridentification of the regions of hypervariability, the two most commonbeing those of Kabat and of Chothia and Lesk. Kabat et al (1991) definethe “complementarity-determining regions” (CDR) based on sequencevariability at the antigen-binding regions of the VH and VL domains.Chothia and Lesk (1987) define the “hypervariable loops” (H or L) basedon the location of the structural loop regions in the VH and VL domains;the numbering for the hypervariable loops is defined as H1: 26-32 or 34;H2: 52-56; and H3: 95-102 (equivalent to CDR3 of Kabat numbering) forVH/VHH domains (Chothia and Lesk, 1987). As these individual schemesdefine CDR and hypervariable loop regions that are adjacent oroverlapping, those of skill in the antibody art often utilize the terms“CDR” and “hypervariable loop” interchangeably, and they may be so usedherein. The CDR amino acids in VH and VL regions are defined hereinaccording to the Kabat numbering system (Kabat et al. 1991).

The region outside of the CDR is referred to as the framework region(FR). The FR provides structural integrity to the variable domain andensures retention of the immunoglobulin fold. This characteristicstructure of antibodies provides a stable scaffold upon whichsubstantial antigen-binding diversity can be explored by the immunesystem to obtain specificity for a broad array of antigens (Padlan etal, 1994). The FR of the variable domain generally show less sequencevariability than the hypervariable regions.

An “antibody fragment” as referred to herein may include any suitableantigen-binding antibody fragment known in the art. For example, anantibody fragment may include, but is by no means limited to Fv (amolecule comprising the V_(L) and V_(H)), single-chain Fv (scFV; amolecule comprising the V_(L) and V_(H) connected with by peptidelinker), Fab, Fab′, F(ab′)₂, single domain antibody (sdAb; moleculescomprising a single variable domain and 3 CDR), and multivalentpresentations of these. The antibody fragment of the present inventionmay be obtained by manipulation of a naturally-occurring antibody (suchas, but not limited to enzymatic digestion), or may be obtained usingrecombinant methods.

By “specific to ricin”, it is meant that the antibody or fragmentthereof of the present invention specifically recognizes and binds toricin. Ricin is a 60-65 kDa glycoprotein derived from beans of thecastor plant (Montanaro et al, 1973). It is a relatively simple toxincomprising a ricin toxin enzymatic-A (RTA) protein and a ricin toxinlectin-B (RTB) protein linked by a disulfide bond. The RTB isresponsible for binding to specific sugar residues on the target cellsurface and allows internalization of ricin by endocytosis, whereas theRTA enzymatically inactivates the ribosome to irreversibly inhibitprotein synthesis. The ricin toxin is one of the most potent toxinsknown for humans.

In a non-limiting example, the isolated or purified antibody or fragmentthereof of the present invention may comprise a variable chaincomprising a CDR L1 of sequence KASQDIKQYIA (SEQ ID NO:1), a CDR L2 ofsequence YTSTLQP (SEQ ID NO:5), and a CDR L3 of sequence LQYDHLYT (SEQID NO:9); and a variable heavy chain comprising CDR H1 of sequence SYWIQ(SEQ ID NO:13), a CDR H2 of sequence EILPGTGNTNYSEKFKG (SEQ ID NO:17),and a CDR H3 of sequence CEGEGYFQAWFAY (SEQ ID NO:21). Alternatively,the isolated or purified antibody or fragment thereof of the presentinvention may comprise variable chain comprising a CDR L1 of sequenceKASQDINNYLR (SEQ ID NO:2), a CDR L2 of sequence RANRLVD (SEQ ID NO:6),and a CDR L3 of sequence LQYDEFPYT (SEQ ID NO:10); and a variable heavychain comprising CDR H1 of sequence EYIIN (SEQ ID NO:14), a CDR H2 ofsequence WFYPGSGDIKYNEKFKD (SEQ ID NO:18), and a CDR H3 of sequenceNGRWDDDYFDY (SEQ ID NO:22). In yet another alternative, the isolated orpurified antibody or fragment thereof of the present invention maycomprise variable chain comprising a CDR L1 of sequence KASQDIKKYIG (SEQID NO:3), a CDR L2 of sequence YTSTLQP (SEQ ID NO:7), and a CDR L3 ofsequence LQYDSLYT (SEQ ID NO:11); and a variable heavy chain comprisingCDR H1 of sequence NYWIE (SEQ ID NO:15), a CDR H2 of sequenceEILPGSGSINYDEKFKG (SEQ ID NO:19), and a CDR H3 of sequence QANRGFDSAWFAY(SEQ ID NO:23). In a further alternative, the the isolated or purifiedantibody or fragment thereof of the present invention may comprisevariable chain comprising a CDR L1 of sequence KASQDVTAAVA (SEQ IDNO:4), a CDR L2 of sequence SASYRYT (SEQ ID NO:8), and a CDR L3 ofsequence QQYYNTPLT (SEQ ID NO:12); and a variable heavy chain comprisingCDR H1 of sequence EHIIN (SEQ ID NO:16), a CDR H2 of sequenceLINPNSGGTNYNQKFKD (SEQ ID NO:20), and a CDR H3 of sequence LRYDAAY (SEQID NO:24).

In one specific, non-limiting example, the isolated or purified antibodyor fragment thereof may comprise the variable light chain sequenceselected from:

(SEQ ID NO: 25) DIQMTQSPSSLSASLGGKVTITCKASQDIKQYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLDPEDIATYYCLQYDHLYTFGGG TKLEIKR;(SEQ ID NO: 27) DIVLTQSPSSMYASLGERVTITCKASQDINNYLRWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGFYSCLQYDEFPYTFGG GTKLEIKR;(SEQ ID NO: 29) DIQMTQSPSSLSAFVGGKVTITCKASQDIKKYIGWYQQKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDSLYTFGGG TKLEIKR;(SEQ ID NO: 31) DIELTQSHKFMSTSVGDRVSITCKASQDVTAAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGSDFTFTISSVQAEDLAVYYCQQYYNTPLTFGA GTKLELKR;and

-   -   a sequence substantially identical thereto

and the variable heavy chain sequence selected from:

(SEQ ID NO: 26) KVQLQESGAELMKPGASVKISCKATGYTFSSYWIQWIKQRPGHGLEWIGEILPGTGNTNYSEKFKGKATFTTDTSSNTAYMHFSSLTSEDSAVYYCSRCEGEGYFQAWFAYWGQGTTVTVSS; (SEQ ID NO: 28)EVQLQESGTGLVKPGASVKLSCKASGYTFTEYIINWVKQRSGQGLEWIGWFYPGSGDIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARNG RWDDDYFDYWGQGTTVTVSS;(SEQ ID NO: 30) KVKLQESGAELMKPGASVKISCKSTGYTFSNYWIEWIKQRPGHGLEWIGEILPGSGSINYDEKFKGKATFTADTSSDTVYMFLSGLTSEDSAVYYCARQANRGFDSAWFAYWGQGTTVTVSS; (SEQ ID NO: 32)QVQLQESGPELVKPGASMKISCKASGYSFTEHIINWVKQTHRENLEWIGLINPNSGGTNYNQKFKDKATLTVDTASNTAYMELLSLTSEDSAVYYCARLR YDAAYWGQGTTVTVSS;and

-   -   a sequence substantially identical thereto.

In another specific, non-limiting example, the isolated or purifiedantibody or fragment thereof may comprise

the variable light chain sequence

(SEQ ID NO: 25) DIQMTQSPSSLSASLGGKVTITCKASQDIKQYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLDPEDIATYYCLQYDHLYTFGGG TKLEIKR

and the variable heavy chain sequence

(SEQ ID NO: 26) KVQLQESGAELMKPGASVKISCKATGYTFSSYWIQWIKQRPGHGLEWIGEILPGTGNTNYSEKFKGKATFTTDTSSNTAYMHFSSLTSEDSAVYYCSRCEGEGYFQAWFAYWGQGTTVTVSS;or

the variable light chain sequence

(SEQ ID NO: 27) DIVLTQSPSSMYASLGERVTITCKASQDINNYLRWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGFYSCLQYDEFPYTFGG GTKLEIKR

and the variable heavy chain sequence

(SEQ ID NO: 28) EVQLQESGTGLVKPGASVKLSCKASGYTFTEYIINWVKQRSGQGLEWIGWFYPGSGDIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARNG RWDDDYFDYWGQGTTVTVSS;or

the variable light chain sequence

(SEQ ID NO: 29) DIQMTQSPSSLSAFVGGKVTITCKASQDIKKYIGWYQQKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDSLYTFGGG TKLEIKR

and the variable heavy chain sequence

(SEQ ID NO: 30) KVKLQESGAELMKPGASVKISCKSTGYTFSNYWIEWIKQRPGHGLEWIGEILPGSGSINYDEKFKGKATFTADTSSDTVYMFLSGLTSEDSAVYYCARQANRGFDSAWFAYWGQGTTVTVSS;or

the variable light chain sequence

(SEQ ID NO: 31) DIELTQSHKFMSTSVGDRVSITCKASQDVTAAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGSDFTFTISSVQAEDLAVYYCQQYYNTPLTFGA GTKLELKR

and the variable heavy chain sequence

(SEQ ID NO: 32) QVQLQESGPELVKPGASMKISCKASGYSFTEHIINWVKQTHRENLEWIGLINPNSGGTNYNQKFKDKATLTVDTASNTAYMELLSLTSEDSAVYYCARLR YDAAYWGQGTTVTVSS;

or a sequence substantially identical thereto.

A substantially identical sequence may comprise one or more conservativeamino acid mutations. It is known in the art that one or moreconservative amino acid mutations to a reference sequence may yield amutant peptide with no substantial change in physiological, chemical, orfunctional properties compared to the reference sequence; in such acase, the reference and mutant sequences would be considered“substantially identical” polypeptides. Conservative amino acid mutationmay include addition, deletion, or substitution of an amino acid; in onenon-limiting example, the conservative amino acid mutation is aconservative amino acid substitution. A conservative amino acidsubstitution is defined herein as the substitution of an amino acidresidue for another amino acid residue with similar chemical properties(e.g. size, charge, or polarity).

A conservative amino acid substitution may substitute a basic, neutral,hydrophobic, or acidic amino acid for another of the same group. By theterm “basic amino acid” it is meant hydrophilic amino acids having aside chain pK value of greater than 7, which are typically positivelycharged at physiological pH. Basic amino acids include histidine (His orH), arginine (Arg or R), and lysine (Lys or K). By the term “neutralamino acid” (also “polar amino acid”), it is meant hydrophilic aminoacids having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms. Polaramino acids include serine (Ser or S), threonine (Thr or T), cysteine(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine(Gln or Q). The term “hydrophobic amino acid” (also “non-polar aminoacid”) is meant to include amino acids exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg (1984). Hydrophobic amino acids include proline (Proor P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val orV), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M),alanine (Ala or A), and glycine (Gly or G). “Acidic amino acid” refersto hydrophilic amino acids having a side chain pK value of less than 7,which are typically negatively charged at physiological pH. Acidic aminoacids include glutamate (Glu or E), and aspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences;it is determined by calculating the percent of residues that are thesame when the two sequences are aligned for maximum correspondencebetween residue positions. Any known method may be used to calculatesequence identity; for example, computer software is available tocalculate sequence identity. Without wishing to be limiting, sequenceidentity can be calculated by software such as NCBI BLAST2 servicemaintained by the Swiss Institute of Bioinformatics (and as found athttp://ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or anyother appropriate software that is known in the art.

The substantially identical sequences of the present invention may be atleast 85% identical; in another example, the substantially identicalsequences may be at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% identical at the amino acid level to sequencesdescribed herein. Importantly, the substantially identical sequencesretain the activity and specificity of the reference sequence. As wouldbe known to one of skill in the art, amino acid residues of an antibody,particularly within the framework regions may be mutated (for example,by conservative substitution) without significantly affecting thefunctional properties of the antibody (antigen recognition and binding).

The isolated or purified antibody or fragment thereof of the presentinvention, and as described herein, may be specific to the ricin toxinlectin-B protein. The isolated or purified antibody or fragment thereofof the present invention may be an IgG.

The antibody or fragment thereof of the present invention alsoencompasses chimeric and humanized constructs based on the variablelight chain or CDR sequences of the antibodies of the present invention.By the term “chimeric”, it is meant that the variable light regions ofthe antibodies of the present invention, as described above, are graftedonto the constant regions (which may include C_(L), C_(H1), C_(H2), andC_(H3)) from a different source. In one specific, non-limiting example,a chimeric construct may comprise the variable light regions of thepresent invention and human constant regions. Methods of preparing suchchimeric constructs are well-known to those of skill in the art (Sun LK, 1987). By the term “humanized”, it is meant that the CDR describedabove may be grafted onto the framework regions of human antibodyfragments (Fv, scFv, Fab, sdAb). The humanized constructs may beprepared using any suitable method know in the art, for example, but notlimited to humanization, CDR grafting, and veneering. Humanization of anantibody or antibody fragment comprises replacing an amino acid in thesequence with its human counterpart, as found in the human consensussequence, without loss of antigen-binding ability or specificity; thisapproach reduces immunogenicity of the antibody or fragment thereof whenintroduced into human subjects. In the process of CDR grafting, one ormore than one of the CDR defined herein may be fused or grafted to ahuman variable region (V_(H), or V_(L)), or to other human antibodyfragment framework regions (Fv, scFv, Fab). In such a case, theconformation of said one or more than one hypervariable loop ispreserved, and the affinity and specificity of the sdAb for its target(i.e., ricin) is also preserved. CDR grafting is known in the art and isdescribed in at least the following: U.S. Pat. No. 6,180,370, U.S. Pat.No. 5,693,761, U.S. Pat. No. 6,054,297, U.S. Pat. No. 5,859,205, andEuropean Patent No. 626390. Veneering, also referred to in the art as“variable region resurfacing”, involves humanizing solvent-exposedpositions of the antibody or fragment; thus, buried non-humanizedresidues, which may be important for CDR conformation, are preservedwhile the potential for immunological reaction against solvent-exposedregions is minimized. Veneering is known in the art and is described inat least the following: U.S. Pat. No. 5,869,619, U.S. Pat. No.5,766,886, U.S. Pat. No. 5,821,123, and European Patent No. 519596.Persons of skill in the art would be amply familiar with methods ofpreparing such humanized antibody fragments.

The antibody or fragment thereof of the present invention may alsocomprise additional sequences to aid in expression, detection, orpurification of a recombinant antibody or fragment thereof. For example,and without wishing to be limiting, the antibody or fragment thereof maycomprise a targeting or signal sequence (for example, but not limited toompA), a detection tag (for example, but not limited to c-Myc,EQKLISEEDL, SEQ ID NO:33), a purification tag (for example, but notlimited to a histidine purification tag, HHHHH, SEQ ID NO:34), or anycombination thereof.

The antibody or fragment thereof of the present invention may also be ina multivalent display. Multimerization may be achieved by any suitablemethod of know in the art. For example, and without wishing to belimiting in any manner, multimerization may be achieved usingself-assembly molecules (Zhang et al, 2004; Merritt & Hol, 1995), forexample as described in WO2003/046560. The described method producespentabodies by expressing a fusion protein comprising the antibody orfragment thereof of the present invention and the pentamerization domainof the B-subunit of an AB₅ toxin family (Nielson et al, 2000); thepentamerization domain assembles into a pentamer, through which amultivalent display of the antibody or fragment thereof is formed. Eachsubunit of the pentamer may be the same or different. Additionally, thepentamerization domain may be linked to the antibody or antibodyfragment using a linker; such a linker should be of sufficient lengthand appropriate composition to provide flexible attachment of the twomolecules, but should not hamper the antigen-binding properties of theantibody. In one non-limiting example, the linker may be the linkerGPGGGSGGGGS (SEQ ID NO:35).

Other forms of multivalent display are also encompassed by the presentinvention. For example, and without wishing to be limiting, the antibodyor fragment thereof may be presented as a dimer, a trimer, or any othersuitable oligomer. This may be achieved by methods known in the art, forexample direct linking connection (Nielsen et al, 2000), c-jun/Fosinteraction (de Kruif et al, 1996), “Knob into holes” interaction(Ridgway et al, 1996).

The present invention also encompasses nucleic acid sequences encodingthe molecules as described herein. The nucleic acid sequence may becodon-optimized. The present invention also encompasses vectorscomprising the nucleic acids as just described.

The present invention additionally comprises hybridoma cells expressingthe antibodies of the present invention. In a specific, non-limitingexample, the present invention provides hybridoma cells A9, B10, D3 andD9, which express antibodies A9, B10, D3 and D9, respectively.

The present invention also provides a composition comprising one or morethan one antibody or fragment thereof, as described herein. Thecomposition may be a vaccine composition. In addition to the one or morethan one antibody or fragment thereof, the composition may comprise apharmaceutically acceptable diluent, excipient, or carrier. The diluent,excipient, or carrier may be any suitable diluent, excipient, or carrierknown in the art, and must be compatible with other ingredients in thecomposition, with the method of delivery of the composition, and mustnot deleterious to the recipient of the composition. The one or morethan one antibody or fragment thereof as described herein may also beformulated in a liposome or other form of encapsulation, using art-knownmethods. The liposome or encapsulation may optionally be formulated fortimed-release; such formulations are well-known in the art.

The composition may be in any suitable form; for example, thecomposition may be provided in suspension form, powder form (forexample, lyophilised), capsule or tablet form. For example, and withoutwishing to be limiting, when the composition is provided in suspensionform, the carrier may comprise water, saline, a suitable buffer, oradditives to improve solubility and/or stability; reconstitution toproduce the suspension is effected in a buffer at a suitable pH toensure the viability of the bacteria. In a specific, non-limitingexample, the pharmaceutically acceptable carrier may be saline. Drypowders may also include additives to improve stability and/or carriersto increase bulk/volume; for example, and without wishing to belimiting, the dry powder composition may comprise sucrose or trehalose.

It would be within the competency of a person of skill in the art toprepare suitable compositions comprising the present compounds.

In yet another alternative, the one or more than one antibody orfragment thereof described herein may be delivered using a gene-therapyapproach. For example, and without wishing to be limiting in any manner,the one or more than one antibody or fragment thereof may be encoded asa DNA vector within defective viruses (such as, but not limited toadenoviruses) for delivery into a subject's cell(s). Methods ofdelivering vaccines or therapeutics in this manner are well-known in theart (Fang J, et al, 2005).

The present invention further provides a method of preventingdeleterious effects caused by ricin exposure or of treating exposure toricin comprising administering one or more than one antibody or fragmentthereof or a composition thereof as described herein to a subject inneed thereof. The subject in need thereof may be any species of mammalthat is susceptible to the effects of ricin; for example, and withoutwishing to be limiting in any manner, the mammal may be a mouse or ahuman.

When using the one or more than one antibody or fragment thereof fortreatment of ricin exposure, the one or more than one antibody orfragment thereof may be administered to the subject up to several hoursfollowing exposure to the ricin toxin to rescue the subject from death.For example, the one or more than one antibody or fragment thereof maybe administered 0, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, or 8 hours following ricin exposure, or any timetherebetween. In specific, non-limiting examples, a single antibody orfragment thereof as described herein may be administered to the subjectbetween 0 and 4 hours following ricin exposure, while a synergisticcombination of antibodies or fragments thereof may be administeredbetween 0 and 8 hours following ricin exposure.

When using the one or more than one antibody or fragment thereof forpreventing deleterious effects caused by ricin exposure (i.e.prophylaxis), the one or more than one antibody or fragment thereof maybe administered to the subject up to several weeks prior to exposure tothe ricin toxin. For example, the one or more than one antibody orfragment thereof may be administered 0, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 weeks prior toricin exposure, or any time therebetween to protect the subject againstricin exposure. In a specific, non-limiting example, a single antibodyor fragment thereof, or a synergistic combination of antibodies orfragments thereof, as described herein may be administered to thesubject between 0 and 9 weeks prior to ricin exposure.

As described above, more than one antibody or fragment thereof of thepresent invention may be combined to provide a synergistic effect withrespect to the ricin-neutralizing activity. For example, and withoutwishing to be limiting in any manner, mAb D9 may be combined with anyone or more of mAb A9, B10, and/or D3 to provide enhanced activityagainst ricin. In one specific example, mAb D9 and B10 may be combinedfor administration. Additionally, mAb D9 may be administered incombination with any prior art antibody to provide a similar synergisticeffect; for example, and without wishing to be limiting in any manner,mAb D9 may be combined with mAb HRF4.

Yet another aspect of the present invention provides a method ofconferring immunity against ricin comprising administering one or morethan one antibody or fragment thereof as described herein, or acomposition thereof. The one or more than one antibody or fragmentthereof or composition comprising same may be administered by anysuitable route know in the art. For example, and not wishing to belimiting, the one or more than one antibody or fragment thereof orcomposition comprising same may be administered subcutaneously,intramuscularly, orally, or by inhalation.

The present invention also provides a method of identifying hybridomasecreting effective anti-ricin antibodies, comprising:

-   -   a) providing hybridoma cells prepared from lymphocytes obtained        from mice immunized against ricin;    -   b) exposing the cells to high amounts of ricin; and    -   c) identifying the cells that survive exposure step b).

In the method as described above, the mice from which the lymphocytesare obtained may have been immunized using stepwise increasing doses ofricin; the stepwise increasing doses may extend into the lethal range.This is contrary to methods commonly used in the art, where sublethalamounts of ricin, ricin toxoid or deglycosylated ricin in adjuvant areused. The hybridoma cells may be prepared from the fusion oflymphocytes, obtained from spleens taken from the immunized mice, and amyeloma cell line; this may be accomplished using any suitable methodknown in the art.

The hybridoma cells are then exposed to high amounts of ricin. Thehybridoma cells may be isolated by dilution into individual containers(such as, but not limited to wells of a sterile microtitre plate)containing sterile cell culture medium. The high amount of ricin used instep b) of the method described above may be any suitable ricin finalconcentration; for example, and without wishing to be limiting in anymanner, the final concentration of ricin may be in the range of 1 to 5ng/ml; for example, the concentration of ricin may be 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, or 5 ng/ml, or any concentration in the range as previouslydefined. In one specific, non-limiting example, the concentration ofricin may be 5 ng/ml. This amount of ricin represents a 25-fold increasein ricin over what has been used in the prior art (Furukawa-Stoffer etal, 1999); without wishing to be bound by theory, this may provide amore rigorous selection of antibody-secreting hybridoma, and allowselection of highly potent neutralizing antibodies.

The method described above may further comprise a step of confirmingthat the hybridoma of step c) survived ricin exposure by assessingsecretion of effective anti-ricin antibodies. This step may be done bymethods known in the art. The antibodies secreted by the hybridoma maybe highly effective in neutralizing ricin.

The method as described above may also include a step of characterizingthe antibody secreted by the hybridoma. The characterization may includeidentification of the antibody isotype, the antibody binding affinityand/or specificity to ricin (using for example, but not limited to ELISAassays or surface plasmon resonance), the antibody activity in in vitro(for example, but not limited to neutralization of ricin in a Vero cellculture) or in vivo (for example but not limited to neutralization ofricin in a mouse model).

Four hybridoma clones were developed and described herein that secretedhigh-titre anti-ricin IgG antibodies. These mAbs have great potential tobe developed as antibody-based therapeutic agents or antibody-gene basedvaccines against ricin. All four mAbs were found to have highricin-neutralization potency both in an in vitro neutrallization assayand an in vivo antibody/ricin co-incubation assay, indicating the stronginhibition of ricin-mediated cell death. Monoclonal antibody D9, foundto be exceptionally active in the mouse assay, was further tested forpost-exposure therapy and pre-exposure prophylaxis against ricin invivo. It protected mice not only hours, but also several weeks (at least6 weeks) before toxin challenge (5×LD₅₀ of ricin), and rescued mice upto 6 hours after poisoning (5×LD₅₀ of ricin); additionally, low amounts(0.5 μg) were therapeutic against high amounts of toxin (1 μg of ricin).Antibody D9 also showed synergistic effects with other anti-ricin mAb,as determined by the in vitro neutralization assay. A dose of 5 μgantibody in a mouse is equivalent to 1.4 mg in a human, which is in thelethal dose range. These results indicate that milligram amounts ofspecific anti-ricin monoclonal antibody in very small volumes (0.1 ml)may be sufficient to protect first responders or treat ricin-exposedcasualties.

Ethical considerations prevent anti-ricin efficacy studies in humans;thus, evaluation of the antibodies or fragments thereof or compositionsof the present invention must be conducted in animal models. The FDA hasdevised a policy, the Animal Rule(http://www.fda.gov/cber/rules/humeffic.htm; also see Federal Register:May 31, 2002 (Volume 67, Number 105, pages 37988-37998)), which permitsapproval of therapeutics or vaccines based on efficacy studies performedexclusively with animal models. The Animal Rule requires that any suchanimal models should mimic the human disease, and that therapeutictreatment or vaccine-elicited protection in animals should predictefficacy in humans. Based on the results in animal models presentedherein and on the FDA's Animal Rule, the antibodies or fragments thereofor compositions of the present invention constitutes an excellentcandidate as an anti-ricin vaccine for both animals and humans.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLE 1 Preparation of Ricin Stock

Because ricin is a possible terrorist biothreat, it is very difficult toobtain from commercial sources. Castor beans were obtained and workingstocks of ricin were prepared. Specifics regarding the source of castorbeans and preparation of the ricin stock cannot be disclosed due tosecurity issues.

Ricin was prepared from castor bean seeds in Defence Research andDevelopment Canada-Suffield. The toxicity of ricin stock was alsodetermined. One LD50 of ricin for mice was determined by the i.p.injection of a series of ricin dilution into mice. The mice wereobserved for 7 days. The amount of ricin for 1×LD50 delivered by thei.p. route for one 20-25 gram female Balb/c mouse was 0.215 μg; 5×LD50was 1.075 μg, or about 1 μg. For 5×LD50 of ricin delivered by the i.p.route, mice died within 1-2 days.

EXAMPLE 2 Creation and Selection of Hybridoma

Mice were used to obtain antibody hybridoma. The mice are kept in asecure BSL-2 area, cared for under the Canadian Council on Animal Care(CCAC) guidelines, and assessed for alternative endpoints.

Groups of 5 BALB/c female mice were injected i.p. with increasingamounts of ricin (0.2, 1.0, 5 and 25×LD₅₀) from Example 1 in 0.1 mlsterile 0.9% saline per mouse. Depending on their recovery (weight gain,a lack of illness symptoms), injections of increasing ricin amounts were2-3 weeks apart. Two weeks after the final dose, the mice were bled bynicking the tail vein with a scalpel while the mouse was in a restraintchamber; blood was collected into a sterile micro centrifuge tube andallowed to clot at room temperature for 30 min. The sample was thencentrifuging at 2300×g for 5 min and the serum was collected; ifrequired, the serum was stored at −20° C. until needed.

ELISA was performed to determine anti-ricin IgG antibody titres.Ninety-six-well ELISA plates (Nunc Maxisorp, Canadian Life Technologies,Burlington, ON) were coated with 100 μl per well of 5 μg/ml ricin incarbonate bicarbonate buffer, pH 9.6, then incubated overnight at 4° C.After blocking with dilute BSA, the plates were incubated with 100 μl ofserum dilutions for 2 hr at room temperature. Anti-ricin antibodies weredetected by incubation with 1:3000 diluted HRP-goat anti-mouse IgG(Caltag Laboratories, Burlingame, Calif.) followed by the addition of atetramethylbenzidine peroxidase substrate (Kirkegaard and PerryLaboratories, Gathersburg, Md.). Absorbance was measured at 615 nm by amicroplate autoreader (Molecular Devices, Sunnyvale, Calif.).

The two mice with the highest titres were sacrificed three days afterthe last booster to collect lymphocytes. These mice were sacrificed bycervical dislocation then the abdomen was swabbed with 70% ethanol andopened with sterile scissor and forceps. Spleens were asepticallydissected from the immunized mice and were ground gently with autoclavedfrosted-glass slides in Dulbecco's Modified Eagle's Medium (DMEM;Invitrogen) then filtered through a wire mesh screen to preparesplenocytes. Hybridomas were produced by fusing the splenocytes with Sp2/0 myeloma cells (ATCC accession number CRL-1581, ATCC, Rockville, Md.)using a Clonacell™-HY Kit (StemCell Technologies, Vancouver, BC),following the manufacturer's instruction and growing these in semisolidmedium with 2.5 ng/ml ricin (10×hybridoma cell culture lethal dose).After 2 weeks, single hybridoma clones were picked up from semisolidmedium, transferred to 96-well tissue culture plates and then grown for1 week in Clonacell Medium E with 5 ng/ml ricin (20×hybridoma cellculture lethal dose) for further selection.

From the surviving clones, the supernatant was removed and assessed byELISA (as described above) for anti-ricin antibodies. The antibodieswere further characterized using a mouse IsoStrip Kit from RocheDiagnostics (Laval, QC) following the manufacturer's instruction. Onlyclones expressing IgG antibodies were further used. Twenty-five clonessurvived this high concentration of toxin and from these, a panel of 4hybridoma clones (A9, B10, D3, D9) with high specific reactivity forricin were identified by ELISA.

EXAMPLE 3 Antibody Purification and Characterization

The four hybridoma clones of Example 2 were cultured and the expressedantibodies were purified and characterized.

Hybridoma clones A9, B10, D3, and D9 were separately cultured in DMEMsupplemented with 10% FBS. Monoclonal antibodies (mAb) were purifiedfrom the cell culture supernatants by Melon Gel purification (Melon GelMonoclonal IgG Purification Kit, Pierce, Rockford, Ill.) according tothe manufacturer's protocol. The supernatant was dialyzed for two 1 hrexchanges in Melon Gel IgG Purification Buffer pH 7.0 and was loadedonto a column containing the Melon Gel resin. After 5 minute incubationwith end-over-end mixing, the purified IgG was collected in theflow-through fraction. All IgG purified samples were aliquoted andstored at minus 20° C. The purity of the mAb was 85-90%, as assessed bySDS-PAGE (data not shown).

The purified mAb were also isotyped using a mouse IsoStrip™ Kit. All themAb showed the same subtype of heavy chain, gamma 1, and the same typeof light chain, kappa. The immunoreactivities of these mAb to the ricinwere investigated by ELISA. All the mAb bound to ricin (FIG. 1) in adose-dependent manner. HRF4 (Furukawa-Stoffer, T. L., 1999) was used asa positive control, showing high activity. Particularly interesting isthe average activity shown by D9 antibody.

Four anti-ricin neutralizing antibody variable sequences were determinedusing PCR with degenerate primers specific for mouse antibodies(Amersham Pharmacia). Briefly, total RNA was isolated from hybridomacell lines (Qiagen RNeasy Plus Mini) and reverse-transcribed withSuperscript II RNase H⁻ (Invitrogen) and an oligo dT primer (12-18 mer)to produce cDNA. Platinum Taq DNA Polymerase High Fidelity (Invitrogen)was used to amplify the ScFv genes, V_(H) and V_(L) with degenerateprimers (Amersham Pharmacia Biotech) for PCR (Eppendorf Mastercylergradient). Distinct bands of about 340 by for V_(H) and about 325 by forV_(L) were detected on a 1.5% agarose gel after PCR and the bands werepurified (Qiagen Gel Extraction) and cloned into Zero Blunt TOPO PCRcloning vector (Invitrogen) for sequencing (Beckman Coulter CEQ 8000Genetic Analyzer).

The amino acid sequences for the variable domains of mAb A9, B10, D3,and D9 are shown below, with CDR regions underlined.

A9 variable light chain (SEQ ID NO: 25)DIQMTQSPSSLSASLGGKVTITCKASQDIKQYIAWYQYKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLDPEDIATYYCLQYDHLYTFGGG TKLEIKRA9 variable heavy chain (SEQ ID NO: 26)KVQLQESGAELMKPGASVKISCKATGYTFSSYWIQWIKQRPGHGLEWIGEILPGTGNTNYSEKFKGKATFTTDTSSNTAYMHFSSLTSEDSAVYYCSRCEGEGYFQAWFAYWGQGTTVTVSS B10 variable light chain (SEQ ID NO: 27)DIVLTQSPSSMYASLGERVTITCKASQDINNYLRWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGFYSCLQYDEFPYTFGG GTKLEIKRB10 variable heavy chain (SEQ ID NO: 28)EVQLQESGTGLVKPGASVKLSCKASGYTFTEYIINWVKQRSGQGLEWIGWFYPGSGDIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARNG RWDDDYFDYWGQGTTVTVSSD3 variable light chain (SEQ ID NO: 29)DIQMTQSPSSLSAFVGGKVTITCKASQDIKKYIGWYQQKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDSLYTFGGG TKLEIKRD3 variable heavy chain (SEQ ID NO: 30)KVKLQESGAELMKPGASVKISCKSTGYTFSNYWIEWIKQRPGHGLEWIGEILPGSGSINYDEKFKGKATFTADTSSDTVYMFLSGLTSEDSAVYYCARQANRGFDSAWFAYWGQGTTVTVSS D9 variable light chain (SEQ ID NO: 31)DIELTQSHKFMSTSVGDRVSITCKASQDVTAAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGSDFTFTISSVQAEDLAVYYCQQYYNTPLTFGA GTKLELKRD9 variable heavy chain (SEQ ID NO: 32)QVQLQESGPELVKPGASMKISCKASGYSFTEHIINWVKQTHRENLEWIGLINPNSGGTNYNQKFKDKATLTVDTASNTAYMELLSLTSEDSAVYYCARLR YDAAYWGQGTTVTVSS

To determine the general specificity of the antibodies, immunoblots wereperformed as follows. Ricin, ricin A-chain and ricin B-chain wereseparated by 10% SDS-PAGE in an X-Cell Sure Lock Mini-Cell apparatus(Invitrogen). The separated proteins were electrophoreticallytransferred onto PVDF membranes (Millipore Corp. Bedford, Mass.) usingMini Trans-Blot system (Bio-Rad Laboratories) with MOPS buffer (50 mMMOPS, 50 mM Tris-base, 0.1% SDS, 1 mM EDTA, pH 7.7, and 10% methanol).Membranes were blocked with Superblock buffer (Fisher ScientificCompany, Canada), followed by 3× washing for 5 min each with PBScontaining 0.05% tween-20 (PBST). The membranes were then incubated withanti-ricin antibodies 1:1000 dilution in Superblock buffer overnight at4° C. Following 3 washes with PBST, the membranes were incubated withIgG-HRP conjugated rabbit anti-mouse antibody (Jackson ImmunoResearchLaboratories) 1:3000 dilution in Superblock buffer at room temperaturefor 1 hr, followed by 3 washes with PBST. The specific binding wasdetected with ECL kit (Amersham Biosciences) and imaged using VersaDoc™5000 system (Bio-Rad Laboratories).

In the SDS-PAGE process above, ricin was disassociated into the highermolecular weight B-chain and lower molecular weight A-chain. All of themAb (A9, B10, D3, and D9) specifically bound to the B-chain. Results forB10, representative of other antibodies, are shown in FIG. 2. As shown,B10 binds to whole ricin (lanes 2) and B-chain (lane 4) but not A-chain(lane 3). All of the present mAb bound to the B-chain, blocked itsability to bind to cell membranes, and so prevented the toxic A-chainfrom entering and killing the cell. This is in contrast to existingantibodies, where most therapeutic candidates are monoclonal antibodieswith binding activity against the toxic A-chain. This is a logicalcourse, as one skilled in the art would seek an antibody that wouldneutralize the toxic part of ricin for effective therapy.

EXAMPLE 4 In Vitro Neutralization Assay

An in vitro neutralization assay involving co-incubation of antibody andtoxin followed by administration to cell culture was used to assess theactivity of the IgG of Example 3.

The amount of antibody was determined by an Easy-Titer Mouse IgG AssayKit (Easy-Titer Mouse IgG Assay Kit, Pierce, Rockford, Ill.) accordingto the manufacturer's protocol. In a microtitre plate, 20 μl of anti-IgGsensitized beads followed by 20 μl of the IgG under investigation wasadded to each well followed by mixing for 5 minutes at room temperature.The plate was then blocked with 100 ul Blocking Buffer for 5 min withmixing and read at an absorbance of 405 nm by a microplate autoreader(Molecular Devices). The antibody concentrations were 4.8 mg/ml for A9,0.68 mg/ml for B10, 1.96 mg/ml for D3 and 1.15 mg/ml for D9.

To determine the activity of a given antibody, it was first diluted inculture media to 10 μg/ml. 200 μl of the diluted antibody was added tothe first well in a microtitre plate column, and 100 μL of culturemedium was added to the other wells of that column. 100 μL wastransferred to the next well in the column to make a 2-fold dilution,this was continued and the last well in the column had 100 μL removed sothat all wells had 100 μL of serially diluted antibody. Ricin wasdiluted in culture media to 300 ng/ml and 50 μL ricin was added to eachwell; the plate was incubated with 5% CO₂ at 37° C. for 2 hours.

Vero cells were maintained in DMEM with 10% FBS (fetal bovine serum) in75 cm² Falcon culture flasks with 5% CO₂ at 37° C., with medium renewalevery 2-3 days. When cells were 60-80% confluent, trypsin was used todetach the cells, and the concentration of cells was estimated bycounting these with a hemocytometer. The cells were diluted to 2×10⁵cells/ml and 50 μl of the cell suspension was added to each well in theabove microtitre plate following the 2-hour incubation of ricin andantibody. The plate was incubated with 5% CO₂ at 37° C. for 2 days.

Following incubation, 20 μL of Alamar Blue (TREK Diagnostic System,Ohio) was added to each well and the plate was incubated with 5% CO₂ at37° C. for 5-6 hours. On a plate reader (Molecular Devices), the platewas read at absorbances of 570 nm and 600 nm, readings were normalizedby subtracting the absorbance reading of wells that did not have cells,and the data point was the average of A_(570nm)÷A_(600nm) of triplicatewells. As would be known to one of skill in the art, Amalar dye diffusesinto dead cells and gives these a high absorbance at 600 nm; if thecells are viable, they will secrete the dye and oxidize Alamar Blue,giving a reduced 600 nm reading and an increased 570 nm reading. Whendividing A_(570nm) by the A_(600nm), larger numbers indicate higherviability of the cells. A standard curve was plotted using readings forwells in the absence of ricin (100% viability), high amounts of ricinand no antibodies (0% survival), and variable amounts of ricin.

The standard curve was used to assess viability of cells in the testwells (ricin co-incubated with mAb). Viability results less than 100%(e.g. 49%) indicate that cells in the test wells (ricin+mAb) were viablebut stopped growing, resulting in low readings compared to control cellsthat continued to grow. Results are shown in Table 1, where it appearedthat B10 mAb performed best in neutralizing ricin in this in vitroassay.

TABLE 1 Relative number of cells surviving 75 ng ricin/mL co-incubatedwith mAb. Viable cells (% of control cells) mAb concentration given 75ng/ml ricin + mAbs (ng/mL) A9 B10 D3 D9 HRF4 5000 65 77 60 106 46 170079 68 40 49 24 560 51 49 33 24 20 190 24 49 21 19 13 62 7 22 14 14 9 219 10 9 15 7 6.9 8 6 7 6 5 2.3 8 7 4 8 7

EXAMPLE 5 In Vivo Neutralization Assay

An in vivo neutralization assay involving antibody administration withricin to mice was used to assess the activity of the IgG of Example 3.

Briefly, different amounts of antibody (from 0.4 to 10 μg/mouse) wereco-incubated with 5×LD₅₀ of ricin (1 hr, 37° C., with gentle inversionmixing every 15 min); the mixture was then injected intraperitoneally(i.p.) into BALB/c female mice. Two antibody gold-standards were used ascontrols: polyclonal goat anti-ricin IgG antibody and mouse mAb HRF4.Results are as shown in Table 2.

TABLE 2 Survival of mice given 5 × LD₅₀ of ricin co-incubated withvarying amounts of antibody. The number of viable mice on each dayfollowing administration is given. Amount Day 1 Antibody (μg) (number)Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 11 Control 0 3 0 — — — — — —Purified 20 3 3 3 3 3 3 3 3 Goat Ab 4 3 1 0 — — — — — 0.8 3 0 — — — — —— HRF4 10 3 3 2 2 1 0 — — 2 3 0 — — — — — — 0.5 5 1 0 — — — — — 0.4 2 0— — — — — — A9 10 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 0.5 5 1 0 — — — — —0.4 3 1 0 — — — — — B10 10 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3 0.5 5 5 4 0— — — — 0.4 3 1 0 — — — — — D3 10 3 3 3 3 3 3 3 3 2 3 3 3 3 1 1 1 1 0.55 5 3 0 — — — — 0.4 3 3 0 — — — — — D9 10 3 3 3 3 3 3 3 3 2 3 3 3 3 3 33 3 0.5 5 5 5 5 5 5 5 5 0.4 3 3 3 1 0 — — —

In in vitro assays, mAb HRF4 was the best binding mAb in ELISA studies(Example 3) and B10 was the best neutralizing antibody in the cellculture assay (Example 4). However, in both in vitro assays, mAb D9appeared unexceptional. Only in the present in vivo mouse modelassessment did D9 show itself to have exceptional merit as a medicalcountermeasure against ricin. Thus, the results of in vitro analysis arenot necessarily indicative of in vivo efficacy of anti-ricin mAb.Surprisingly, 0.5 μg of D9 antibody was effective in protecting micefrom 5×LD₅₀ (1 μg ricin). At this level of efficacy, mAb D9 surpassesthe activity of all other antibodies reported to date.

It is worth noting that all antibodies of the present inventionout-performed the previous gold standard monoclonal antibody, HRF4, aswell as the goat anti-ricin polyclonal antibodies.

EXAMPLE 6 Efficacy of Antibody given before or after Ricin Exposure

Existing publications suggest ricin enters a mammalian cell after onlyabout 30 minutes, and that not much can be done to rescue a casualty oranimal beyond this time. Also, current literature suggests anti-ricinantibodies can be administered hours to a few days before ricinpoisoning to protect mice. In this Example, the survival of miceadministered the antibodies of Example 3 at various time-points prior toor following ricin exposure is assessed.

Antibody administration following ricin exposure: The mice were assessedusing the in vivo neutralization assay as described in Example 5, exceptthat 5 μg of antibody (A9, B10, D3, D9, HRF4 (positive control)) wasadministered 1, 2, 4, or 6 hours following ricin exposure, or saline wasadministered at 1 hour following ricin exposure (negative control). Eachexperimental group comprised 4 mice. Results are shown in Table 3.

TABLE 3 Survival of mice administered antibody at varying time-pointsafter receiving 5 × LD₅₀ of ricin. The number of viable mice on each dayfollowing administration of antibody is given. Time points Day 1 Day 2Day 3 Day 4 Day 5 Day 6 Day 7 Control 1 hr 4 1 0 — — — — HRF4 1 hr 4 2 0— — — — 5 μg per 2 hr 3 1 0 — — — — mouse 4 hr 4 2 0 — — — — 6 hr 3 0 —— — — — A9 1 hr 4 4 4 4 4 4 4 5 μg per 2 hr 4 4 2 2 0 — — mouse 4 hr 4 43 2 0 — — 6 hr 4 3 1 1 0 — — B10 1 hr 4 4 4 4 4 4 4 5 μg per 2 hr 4 4 44 4 4 4 mouse 4 hr 4 4 4 2 2 2 2 6 hr 4 3 3 0 — — — D3 1 hr 4 3 3 3 3 33 5 μg per 2 hr 4 4 4 4 2 2 2 mouse 4 hr 4 4 2 2 0 — — 6 hr 4 4 2 0 — —— D9 1 hr 4 4 4 4 4 4 4 5 μg per 2 hr 4 4 4 4 4 4 4 mouse 4 hr 4 4 4 4 44 4 6 hr 4 4 4 4 4 4 4 8 hr 3 2 0 — — — —

All antibodies of the present invention were capable of rescuing micewhen antibody was administered 1-2 hours following ricin exposure. Infact, and as in Example 5, the present antibodies out-performed bothHRF4 and the goat polyclonal antibodies. Table 3 shows conclusively thatantibodies, especially D9, can be given several hours after ricinpoisoning to rescue mice.

Antibody administration prior to ricin exposure: Due to superiority ofthe D9 antibody and to reduce the amount of animals required forexperimentation, this portion of testing was done using only D9 mAb. Themice were assessed using the in vivo neutralization assay as describedin Example 5, except that 5 μg D9 mAb was administered 1, 7, 14, 28, or42 days prior to ricin exposure, or no antibody was administered priorto ricin exposure (negative control). Each experimental group comprised4 mice. Results are shown in Table 4.

TABLE 4 Survival of mice administered D9 mAb at varying time-pointsprior to receiving 5 × LD₅₀ of ricin. The number of surviving mice 7days following administration of ricin is given. Time point SurvivalNegative control 0* D9  1 day 4 (100%)  7 days 4 (100%) 14 days 4 (100%)28 days 4 (100%) 42 days 4 (100%) *all died or had to be terminatedafter 1 day

Table 4 shows that, aside from some minor temporary weight loss (datanot shown), no deaths were observed when D9 antibody was given 1, 7, 14,28 or 42 days before mice were administered 5×LD₅₀ of ricin. Based onprevious results, it can be hypothesized that mAb A9, B10, and D3 wouldbe similarly, if only slightly less, effective.

Given that there is some clearance or turnover with time, the questionof the amount of time necessary for the 5 μg of administered antibody toattain levels below the protective amount of 0.5 μg antibody (determinedin Example 5) within the mouse was addressed. To conserve on the use ofmice, an extrapolation was done by assessing the amount of D9 antibodyin mouse blood over different time points. Mice were given 5 μg of D9antibody each, and each week a group of mice was bled via that tail vein(see Example 2), the sera collected and the amount of D9 antibody inthat sera assessed by ELISA quantitation (see Example 2). Results areshown in FIG. 3, where the half life of the anti-ricin D9 antibody wasestimated at 18.5 days in the mice. From this data, it can beextrapolated that after 6 weeks the amount of D9 per mouse would beabout 1 μg, above the 0.5 μg minimum. This large window for protectionis understandable given that D9 is a mouse antibody circulating withinmice. A humanized anti-ricin monoclonal antibody based on the antibodiesof the present invention, circulating in a human, may have a similarlylong half-life.

It is difficult to compare efficacy of different anti-ricin antibodiespreviously reported in the literature using different experimentalsettings, such as different antibody administration routes, differentanimal models, and so on. However, two reports appear to have experimentsettings were similar to the present Examples. One report showed thatanti-ricin antibody CD12 or R70, at the dose of 5, 10, 20, or 40μg/mouse could protect mice against 5×LD₅₀ of ricin challenge when theantibody was administered 24 h before ricin challenge (Neal L M, et al,2010). The second report showed that 100 μg/mouse anti-ricin antibody4C13 could rescue mice 30 minutes after ricin challenge (10×LD₅₀; Guo J,et al, 2006). In contrast, the present Examples show that administrationof 5 μg/mouse of D9 antibodycan protect mice for at least 6 weeks beforericin challenge (5×LD50) or can rescue mice 6 hours after ricinchallenge (5×LD50).

Example 7 Synergistic Effect of D9 mAb

Combinations of the antibodies of Example 3 were assessed to assess thepresence of synergistic activities.

The mice were assessed using the in vitro neutralization assay asdescribed in Example 4, except that 1:1 ratio mixtures of antibody (A9,B10, D3, D9, HRF4) were used, at a total concentration of 313 ng/ml.Antibody alone was also used, at a concentration of 156 ng/ml. Resultsare shown in FIG. 4.

A very large set of data was generated but in summation, no matter whichantibody was used, D9 had a helper effect, especially for B10. If theeffect of the antibody combination was simply additive, the results forthe antibody alone and the combination should be equivalent. FIG. 4shows that the values for cell survival were far higher when D9 wasadded to any of the other mAb.

To evaluate the synergistic effect in vivo, the effect of administeringthe combination of D9 and B10 at various time-points following ricinexposure was assessed. This was performed according to the methoddescribed in Example 6, except that 0.5 μg of D9 mAb and 0.5 μg of B10mAb, or 5 μg of D9 mAb and 5 μg of B10 mAb. Synergism was furtherobserved when either 0.5 μg of D9 mAb and 0.5 μg of B10 mAb, 5 μg of D9mAb and 5 μg of B10 mAb, or saline were administered to mice 8 hoursafter ricin poisoning (n=4, each group). Results are shown in Table 5.

In Example 6, the best candidate, D9 mAb, did not differ from salinecontrols when given 8 hours after ricin poisoning (Table 3); if thecombination of D9 and B10 had any synergistic additional effect, itwould be seen at this time point. The present results (Table 5) showedthat the combination of antibodies either prevented death or extendedthe time of death. Specifically, 1 of 4 mice survived when administered0.5 μg each D9 and B10, while life was extended a few days for miceadministered 5 μg each D9 and B10. The extended time to death isencouraging, as it may open a window of opportunity for casualties toreceive supportive care and increased survival following ricin exposure.

TABLE 5 Survival of mice that administered mAb therapy 8 hours followingadministration of 5 × LD₅₀ of ricin. The number of viable mice on eachday following administration of antibody is given. Number of survivingmice from a group of 4 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Ricin +saline 4 0 — — — — — 0.5 μg of D9 mAb + 4 2 1 1 1 1 1 0.5 μg of B10 mAb5 μg of D9 mAb + 4 4 3 2 0 — — 5 μg of B10 mAb

EXAMPLE 8 Chimeric Construct of Anti-Ricin Antibodies

Chimeric constructs of antibodies of Example 3 were prepared. Here, theterm “chimeric” is used to define an antibody where the mouse antibody'sconstant region is replaced with a human constant region.

Briefly, variable regions of heavy and light chains for B10 and D9 weregrafted onto human gamma 1 heavy chain constant region and kappa 1 lightchain constant region, respectively, to assemble the whole chimericantibody genes, resulting in chimeric B10 and D9.

The chimeric antibody DNA sequence (2 kb) was synthesized as follows. Alight chain leader sequence was upstream from the light chain, followedby a foot-and-mouth disease virus 2A self-cleavage linker encodingAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 36) before the heavy chain. Thewhole DNA sequence flanked by Kpn I and Hind III was synthesized byGenScript Corporation (Scotch Plaines, N.J.) and cloned into pUC57vector, resulting in pUC57-chimeric B10 or D9. A recombinant adenovirusvector expressing chimeric B10 or chimeric D9 was constructed usingAdEasy system (Qbiogene, Carlsbad, Calif.) according to themanufacturers protocol. Briefly, Kpn I-Hind III fragment ofpUC57-chimeric B10 or pUC57-chimeric D9 was ligated to a Kpn I-HindIII-digested pShuttle-CMV vector. The resulting pShuttle construct wasco-transformed with the pAdEasy-1 vector into E. coli BJ5183 cells toproduce recombinant adenoviral genomic construct for hu1A4A1IgG1protein. The recombinant adenoviral construct, pAd-chimeric B10 or D9was linearized with Pac I and transfected into HEK 293 cells (ATCC)cultured in Dulbecco's Modified Eagle's Medium supplemented with 5%fetal bovine serum (FBS) for amplification and then the amplifiedadenovirus was purified by a chromatographic method.

To express chimeric B10 or chimeric D9, HEK 293 cells were infected withthe recombinant adenovirus pAd-chimeric B10 or pAd-chimeric D9 at amultiplicity of infection of 1. The infected cells were cultured for oneweek and the culture supernatant was harvested. The expressed chimericB10 or chimeric D9 was purified using protein L agarose gel from PierceBiotechnology (Rockford, Ill.). Briefly, culture supernatant wasdialyzed against phosphate buffer saline (PBS) (Sigma-Aldrich) for 12 hrthen concentrated using PEG (Sigma-Aldrich) to less than 50 ml. Theconcentrated sample was incubated with 2 ml protein L agarose gel at 4°C. for 1 hr. The gel and supernatant mixture was then loaded onto anempty column, which was subsequently washed with binding buffer. Boundchimeric B10 or chimeric D9 was eluted with elution buffer. The elutedAb was further desalted using excellulose column (Pierce Biotechnology)then concentrated by Centricon YM-30 (Millipore Corp., Bedford, Mass.).

The embodiments and examples described herein are illustrative and arenot meant to limit the scope of the invention as claimed. Variations ofthe foregoing embodiments, including alternatives, modifications andequivalents, are intended by the inventors to be encompassed by theclaims. Furthermore, the discussed combination of features might not benecessary for the inventive solution.

EXAMPLE 9 Humanization of Antibodies

Molecular Modeling and Structural Analysis of D9 Fv

Different approaches have been developed to humanize murine antibodiesin order to reduce the antigenicity of murine antibodies in humans.Despite the development of molecular display technologies and transgenicanimals for the generation of fully human antibodies, CDR grafting totransfer all murine antibody CDRs onto the human antibody FRs remains anattractive and proven strategy for overcoming therapeutic deficienciesof murine antibodies.

CDR-grafted antibodies tend to lose the parental binding affinity. Thekey for CDR-grafted antibodies to remain the parental binding affinitylies in the preservation of the murine CDR conformation in the humanizedantibody for antigen binding. The CDR conformation is mainly dependenton CDR canonical structures determined by a few canonical conservedresidues located in CDRs and FRs. Furthermore, some key resides in FRsfine-tune the CDR conformation. They include vernier zone resides,forming a layer underlying the CDRs and interchain packing resides,pairing of CDRs of VH and VL. In order to determine those key FRresidues, the molecular model of D9 variable region was establishedthrough PIGS (http://www.biocomputing.it/pigs), a web server for theautomatic modeling of immunoglobulin variable domains based on the mosthomologous antibody VH (2NR6), sharing 86% identity and VL (1MLB),sharing 70% identity with the corresponding VH and VL of D9 in thedatabase of known immunoglobulin structure. Three D structure of D9 wasthen visualized using a pdb molecular visualisation programme(Deepview), the vernier zone residues located in 5 Å of the CDRs and theinterchain packing resides located in 5A of VH-VL interface wereidentified shown in FIG. 5.

Humanization of D9 mAb

There are two sources of human antibody sequences: mature and germline.The latter has two advantages over the former as FR donors for murineCDR grafting. The first is less immunogenic, unlike the mature sequencesthat carry somatic mutations for affinity maturation generated by randomprocesses, resulting in potential immunogenicity. The other is moreflexible, resulting in more compatibility between murine CDRs and humanFRs. Therefore, human germline antibody sequences have increasingly beenutilized as source of FR donors.

In order to select germline human antibody VH, JH and VL, JL candidatesas FR donors for D9 humanization, D9 CDR canonical structures weredetermined first based on identification of unique residues both in CDRsand FRs, and then formed a shortlist of germline human antibody VH andVL candidates. Those had the same or closely related canonicalstructures as D9 to ensure that the human antibody FR supports the mouseCDR canonical structures. Next, within the shortlist of germline humanantibody VH and VL, those with highest homology CDRs and key residues inFR 1-3 were chosen as FR 1-3 donors. Human JH and JL were chosen basedon highest homology to CDR3 and key residues in FR 4. Consequently,human VH gene 1-18, JH gene 6 were selected as FR donors forhumanization of D9 VH; human Vk gene O12 and Jk gene 4 were selected asFR donors for humanization of D9 VL domain shown in FIG. 5. Seventy-five% of the key FR resides of D9 was the same as human donor antibodies.Another 22% were different between murine D9 and human donors, but thesewere conservative substitutions in the same groups of amino acids, suchas S=>T (polar, non-aromatic with hydroxyl R-groups), K=>R or E=>Q orQ=>K (polar, hydrophilic), I=>M or A=>V or L=>M (non-polar,hydrophobic), H=>Y (polar, aromatic), V=>T (β-C containing branch), S=>A(tiny), D=>S (polar). The remaining 3% (2 residues) were not conserved,these being VH44 (mouse N versus human G) and VH82a (mouse L versushuman R). Most importantly, VH44-N was an unusual interchain packingresidue. Only 0.3% VH have N in position 44, indicating it came fromsomatic mutation, which might enhance antibody binding. VH82a-L was avernier zone residue. Advantageously, molecular modeling revealed bothof these as not solvent accessible, indicating these are not located onthe surface of Fv and might not elicit an immune response in human.Therefore, when the CDRs of D9 were grafted onto the donor humanantibody FRs, VH44-N and VH82a-L were kept in the humanized D9 (hD9).

Expression and Purification of hD9

The VH of hD9 was further grafted onto the human gamma 1 heavy chain CHsto form a complete heavy chain, while the VL was grafted onto the humankappa 1 light chain CL to form a whole humanized light chain (FIG. 6).Furthermore, a foot-and-mouth-disease virus-derived 2A self-processingsequence was introduced between heavy and light chain DNA sequences toexpress a full-length antibody from a single open reading frame drivenby a single promoter in an adenoviral vector. To get the expressed hD9to be secreted to culture media, a leader sequence was added upstream tothe VH and VL respectively. The whole DNA sequence including the humanantibody kappa light chain O12 leader sequence, the humanized lightchain (VL+CL), 2A linker, 1-18 heavy chain leader sequence, andhumanized heavy chain (VH+CH1+CH2+CH3), around 2 kb was synthesized asshown in FIG. 6 and then cloned into an adenoviral vector forexpression.

After the recombinant hD9 was expressed in mammalian cells and purifiedusing an ImmunoPure Protein (L) agarose column, the purified product wassubjected to 10% SDS-PAGE. One obvious band of about 150 kDa innon-reducing conditions and two clear bands of about 50 kDa (heavychain) and about 25 kDa (light chain) in reducing conditions (cleavageof disulfide bridges) were observed (FIG. 7), indicating the heavy andlight chain of the recombinant hD9 was cleaved completely and properlydimerized.

Affinity Characterization of hD9

To evaluate the binding affinity of hD9, a surface plasmon resonance(SPR) biosensor was used. Ricin was captured on a biosensor chip,various concentrations of hD9 or D9 were passed through the flow cell,and the binding kinetics was recorded. The kinetic rate constants k_(on)and k_(off) were calculated from the ascending rate of resonance unitsduring association and the descending rate during dissociation. The KDof hD9 or D9 for ricin was determined from the ratio of k_(off)/k_(on).As shown in Table 6, hD9 had high affinity to ricin with KDs of 1.63 nM,even higher than D9 (2.56 nM), indicating humanization of D9 issuccessful.

TABLE 6 Comparison of kinetic constants binding to ricin between of D9and hD9. Antibody K_(on) (M⁻¹S⁻¹) K_(off) (S⁻¹) KD (nM) hD9  4.19 × 10⁵6.8 × 10⁻⁴ 1.63 D9 1.835 × 10⁵ 4.7 × 10⁻⁴ 2.56

Efficacy Evaluation of hD9

To evaluate hD9 efficacy in vivo, ricin was given at the dose of 5×LD50to mice by i.p route. hD9 at the dose of 5 μg was administered by thei.p. route at 2, 4, 6 hr after ricin challenge. hD9 could rescue mice upto 6 hr post-challenge, allowing 50% mouse survival (Table 7),comparable to D9, which showed 100% protection up to 6 hrpost-challenge. This humanized D9 has potential use for prophylactic ortherapeutic purposes against ricin poisoning.

TABLE 7 Survival of mice administered hD9 at varying time points afterreceived 5 × LD50 of ricin. The number of viable mice on each dayfollowing administration of antibody is given. Time points Day 1 Day 2Day 3 Day 4 Day 5 Day 6 Day 7 hD9 5 μg per 2 hr 8 8 8 8 8 8 8 mouse 4 hr8 8 8 8 8 8 8 6 hr 8 8 8 8 8 4 4

REFERENCES

All patents, patent applications and publications referred to herein andthroughout the application are hereby incorporated by reference in theirentirety.

Audi J, Belson M, Patel M, Schier J, Osterloh J. Ricin poisoning: acomprehensive review. JAMA. 2005 Nov. 9; 294(18):2342-51.

Burnett J C, Henchal E A, Schmaljohn A L, Bavari S. The evolving fieldof biodefence: therapeutic developments and diagnostics. Nat Rev DrugDiscov. 2005 April; 4(4):281-97.

Chothia C, Lesk A M. Canonical structures for the hypervariable regionsof immunoglobulins. J Mol Biol. 1987; 196(4):901-17.

de Kruif, J. & Logtenberg, T. Leucine zipper dimerized bivalent andbispecific scFv antibodies from a semi-synthetic antibody phage displaylibrary. J Biol Chem 271, 7630-7634 (1996).

Eisenberg, D.; E. Schwarz; M. Komaromy & R. Wall (1984) Analysis ofmembrane and surface protein sequences with the hydrophobic moment plot.J Mol Biol, 179, 125-142

Fang J, Qian J J, Yi S, Harding T C, Tu G H, VanRoey M, Jooss K. Stableantibody expression at therapeutic levels using the 2A peptide.NatBiotechnol. 2005 May; 23(5):584-90.

Foxwell B M, Detre S I, Donovan T A, Thorpe P E. The use of anti-ricinantibodies to protect mice intoxicated with ricin. Toxicology. 1985January; 34(1):79-88.

Furukawa-Stoffer, T. L., Mah, D. C. W., Cherwonogrodzky, J. W.,Weselake, R. J. 1999. A novel biological-based assay for the screeningof neutralizing antibodies to ricin. Hybridoma 18:505-511.

Guo J, Shen B, Sun Y, Yu M, Hu M. A novel neutralizing monoclonalantibody against both ricin toxin A and ricin toxin B, and applicationof a rapid sandwich enzyme-linked immunosorbent assay. Hybridoma. 2006August; 25(4):225-9

Hewetson J F, Rivera V R, Creasia D A, Lemley P V, Rippy M K, Poli M A.Protection of mice from inhaled ricin by vaccination with ricin or bypassive treatment with heterologous antibody. Vaccine. 1993;11(7):743-6.

Kabat E A, Wu T T. Identical V region amino acid sequences and segmentsof sequences in antibodies of different specificities. Relativecontributions of VH and VL genes, minigenes, andcomplementarity-determining regions to binding of antibody-combiningsites. J Immunol. 1991; 147:1709-19.

Lin J Y, Liu S Y. Studies on the antitumor lectins isolated from theseeds of Ricinus communis (castor bean). Toxicon. 1986; 24(8):757-65.

Merritt, E. A. & Hol, W. G. AB5 toxins. Current opinion in structuralbiology 5, 165-171 (1995).

Miller D J, Ravikumar K, Shen H, Suh J K, Kerwin S M, Robertus J D.Structure-based design and characterization of novel platforms for ricinand shiga toxin inhibition. J Med Chem. 2002 Jan. 3; 45(1):90-8.

Montanaro L, Sperti S, Stirpe F. Inhibition by ricin of proteinsynthesis in vitro. Ribosomes as the target of the toxin. Biochem J.1973 November; 136(3):677-83.

Neal L M, O'Hara J, Brey R N 3rd, Mantis N J. A monoclonalimmunoglobulin G antibody directed against an immunodominant linearepitope on the ricin A chain confers systemic and mucosal immunity toricin. Infect Immun. 2010 January; 78(1):552-61. Epub 2009 Oct. 26.

Nielsen, U. B., Adams, G. P., Weiner, L. M. & Marks, J. D. Targeting ofbivalent anti-ErbB2 diabody antibody fragments to tumor cells isindependent of the intrinsic antibody affinity. Cancer Research 60,6434-6440 (2000).

Padlan, E. A. Anatomy of the antibody molecule. Molecular immunology 31,169-217 (1994).

Ridgway, J. B., Presta, L. G. & Carter, P. ‘Knobs-into-holes’engineering of antibody CH3 domains for heavy chain heterodimerization.Protein Eng 9, 617-621 (1996).

Smallshaw J E, Richardson J A, Vitetta E S. RiVax, a recombinant ricinsubunit vaccine, protects mice against ricin delivered by gavage oraerosol. Vaccine. 2007 Oct. 16; 25(42):7459-69. Epub 2007 Aug. 30.

Sun L K, Curtis P, Rakowicz-Szulczynska E, Ghrayeb J, Chang N, MorrisonS L, Koprowski H. Chimeric antibody with human constant regions andmouse variable regions directed against carcinoma-associated antigen17-1A. Proc Natl Acad Sci USA. 1987 January; 84(1):214-8.

Zhang, J. et al. Pentamerization of single-domain antibodies from phagelibraries: a novel strategy for the rapid generation of high-avidityantibody reagents. J Mol Biol 335, 49-56 (2004).

International PCT Publication No. WO2003/046560, U.S. Pat. No.6,180,370, U.S. Pat. No. 5,693,761, U.S. Pat. No. 6,054,297, U.S. Pat.No. 5,859,205, U.S. Pat. No. 5,869,619, U.S. Pat. No. 5,766,886, U.S.Pat. No. 5,821,123, European Patent No. 519596 and European Patent No.626390.

1. An isolated or purified antibody or fragment thereof comprising avariable chain comprising a CDR L1 of sequence KASQDINNYLR (SEQ IDNO:2), a CDR L2 of sequence RANRLVD (SEQ ID NO:6), and a CDR L3 ofsequence LQYDEFPYT (SEQ ID NO:10); and a variable heavy chain comprisingCDR H1 of sequence EYIIN (SEQ ID NO:14), a CDR H2 of sequenceWFYPGSGDIKYNEKFKD (SEQ ID NO:18), and a CDR H3 of sequence NGRWDDDYFDY(SEQ ID NO:22), wherein the isolated or purified antibody or fragmentthereof is specific for the ricin toxin lectin-B protein.
 2. Theisolated or purified antibody or fragment thereof of claim 1,comprising: the variable light chain sequence: (SEQ ID NO: 27)DIVLTQSPSSMYASLGERVTITCKASQDINNYLRWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGFYSCLQYDEFPYTFGG GTKLEIKR

and the variable heavy chain sequence: (SEQ ID NO: 28)EVQLQESGTGLVKPGASVKLSCKASGYTFTEYIINVWKQRSGQGLEWIGWFYPGSGDIKYNEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARNG RWDDDYFDYWGQGTTVTVSS,

or a sequence at least 95% identical thereto.
 3. The isolated orpurified antibody or fragment thereof of claim 5, wherein the isolatedor purified antibody or fragment thereof is humanized.
 4. The isolatedor purified antibody or fragment thereof of claim 5, wherein theantibody is an IgG.
 5. A composition comprising one or more than oneantibody or fragment thereof of claim 1 and a pharmaceuticallyacceptable diluent, excipient, or carrier.
 6. The composition of claim5, wherein the composition is a vaccine composition.
 7. A method ofpreventing deleterious effects caused by ricin exposure or of treatingexposure to ricin, comprising administering one or more than oneantibody or fragment thereof of claim 5 or the composition of claim 5 toa subject in need thereof.
 8. The method of claim 7, wherein the subjectis a mouse or a human.
 9. The method of claim 7, wherein the one or morethan one antibody or fragment thereof is administered to the subjectseveral hours following exposure to the ricin toxin to treat ricinexposure.
 10. The method of claim 7, wherein the one or more than oneantibody or fragment thereof is administered to the subject severalweeks prior to exposure to the ricin toxin to protect the subjectagainst ricin exposure.
 11. The method of claim 7, further comprisingadministering a second antibody or fragment thereof said second antibodyor fragment thereof comprising the variable light chain sequence:(SEQ ID NO: 31) DIELTQSHKFMSTSVGDRVSITCKASQDVTAAVAWYQQKPGQSPKLLIHSASYRYTGVPDRFTGSGSGSDFTFTISSVQAEDLAVYYCQQYYNTPLTFGA GTKLELKR

and the variable heavy chain sequence: (SEQ ID NO: 32)QVQLQESGPELVKPGASMKISCKASGYSFTEHIINVWKQTHRENLEWIGLINPNSGGTNYNQKFKDKATLTVDTASNTAYMELLSLTSEDSAVYYCARLR YDAAYWGQGTTVTVSS,

or a sequence at least 95% identical thereto.
 12. The method of claim11, wherein at least one of the antibodies or fragments thereof ishumanized.
 13. A method of conferring immunity against ricin comprisingadministering one or more than one antibody or fragment thereof of claim1, or a composition of claim 5 to a subject in need thereof.
 14. Themethod of claim 13, wherein the one or more than one antibody orfragment thereof is administered to the subject 1 day to several weeksprior to exposure to the ricin toxin to protect the subject againstricin exposure.